Copper resistant, fengycin-producing Bacillus mojavensis strain for controlling vegetable pathogens, its use and compositions containing it

ABSTRACT

A biological material is disclosed for exerting antagonism against vegetable pathogens, which contains  Bacillus mojavensis  R3B mutant strain deposited under # NCAIM (P) B 001389 according to the Budapest Treaty. The biological material according to the invention is an effective antagonist against the pathogens of vegetables, preferably tomato, pepper, lettuce and/or cabbage, in particular against the pathogens selected from the group of  Xanthomonas vesicatoria, Pseudomonas syringae  and  Clavibacter michiganensis  vegetable pathogen bacteria and  Pythium debaryanum, Phytophthora infestans, Alternaria alternata  and  Fusarium oxysporum  vegetable pathogen fungi. Furthermore, the invention relates to a composition comprising the biological material according to the invention and optionally a copper-containing pesticide and a process for controlling vegetable pathogens, furthermore use of the biological material or composition according to the invention for the protection of vegetables, preferably tomato, pepper, lettuce and/or cabbage.

This is the national stage of International ApplicationPCT/HU2012/000084, filed Aug. 30, 2012.

BRIEF DESCRIPTION OF THE INVENTION

The invention relates to a biological material for exerting antagonismagainst vegetable pathogens, which contains Bacillus mojavensis R3Bmutant strain deposited on Aug. 8, 2011 at the National Collection ofAgricultural and Industrial Microorganisms, Somlói ut 14-16, 1118Budapest, Hungary, under accession number NCAIM (P) B 001389 accordingto the Budapest Treaty. The biological material according to theinvention is an effective antagonist against the pathogens ofvegetables, preferably tomato, pepper, lettuce and/or cabbage, inparticular against the pathogens selected from the group of Xanthomonasvesicatoria, Pseudomonas syringae and Clavibacter michiganensisvegetable pathogen bacteria and Pythium debaryanum, Phytophthorainfestans, Alternaria alternata and Fusarium oxysporum vegetablepathogen fungi. Furthermore, the invention relates to a compositioncomprising the biological material according to the invention andoptionally a copper-containing pesticide and a process for controllingvegetable pathogens, furthermore use of the biological material orcomposition according to the invention for the protection of vegetables,preferably tomato, pepper, lettuce and/or cabbage.

BACKGROUND OF THE INVENTION

The effective pest control is part of the intensive vegetable productiontechnologies, however, the expectations of the consumer markets,furthermore the efforts to improve the food safety called for theminimization of pesticides, and replacing them by biological methods. Ina closed area production the entire biological control has been achievedagainst insect pests, however, the biological control of bacteria andfungi is still to be elaborated.

Nowadays there is an increasing need in the fields of vegetableproduction and sales for bioproducts containing no residues of chemicalpesticides. Among the effective chemical pesticides only the inorganiccopper-compositions are allowed in the course of production ofbioproducts. The number of copper resistant mutants among the vegetablepathogen microorganisms has increased a lot in the last couple of years,thus these copper containing agents alone are insufficient to achieveeffective and reliable pest control. One possible solution to overcomethis problem is to effect an integrated pest control using coppercontaining agents and biocontol compositions. Biocontrol products mayalso be used alone, but in such cases the highest efficiency of the bestcompositions is still under 50 percent.

The majority of the antibacterial or antifungal biocontrol productspresently available in the world market contain one component as activeagent, which is a bacterium or fungi possessing antagonist and/orparasite features. The Bacillus strain has long been used for vegetablepest control purposes. The compositions sold are especially effectiveagainst vegetable pathogen fungi. The compositions already beencommercialized and patented comprise mainly the strains of Bacillussubtilis and Bacillus amiloliquefaciens species. Their effectiveness isexplained by different peptide antibiotics, secreted outside of the cellwalls, which are effective mainly against fungi: surfactin, iturin,fengycin, bacillomycin, mycosubtilin. Recently it has been suggestedthat their effectiveness is enhanced by extracellular enzymesdecomposing the cell walls and/or the membrane of the cytoplasm of thepathogen microorganism: proteases, kitinases and lipases. Besides,several independent investigations have proved that of the secretedantibiotics mainly fengycin is responsible for exerting of the inducedresistance response in the treated plants.

THE STATE OF THE ART

U.S. Pat. No. 2010092442 relates to a process to induce an acquiredsystemic resistance of plants against infections. The method is based onthe utilization of a composition comprising bacteria belonging to theBacillus strain, as a consequence of which the plant produces protectingproteins. The applied bacteria are the isolate of Bacillus mojavensis203-7-, and the isolate of Bacillus mycoides BmJ. The drawback of theBacillus mojavensis isolate are on one hand that the application of thegiven Bacillus mojavensis 203-7 isolate and the antibacterial agenttogether is not proposed, on the other hand, the isolated strain is notcopper resistant, thus the combined application thereof withcopper-containing pesticides would not achieve the expected pest controleffect. Furthermore, the Bacillus mojavensis 203-7 isolate disclosed inthe patent has no fengycin hyper-producing properties.

International patent application No. WO9724433 discloses biocontrolmethods and microorganisms, which are capable of controlling plantdiseases of fungi origin. The microorganisms useful for biocontrolpurposes are novel endophyte symbiotic Enterobacter cloacae strains.Said strains are suitable for passing useful genetic products into theplants. The used Enterobacter cloacae strains are rifampicin resistant.The Enterobacter cloacae strains according to the patent application(deposited under No. ATCC55732) as analysed their 16S rRNS gene sequencehave proven to be Bacillus mojavensis strains. The shortcoming of theisolate according to the patent application is that it has no copperresistance, therefore its co-application with copper containingpesticides is likely to be discouraged.

International patent application No. WO9909834 discloses compositionsand methods for the treatment of arable soil, which are capable ofimproving the growth of plants and the results of reaping. Thecomposition is based on pesticide resistant microorganism strains. Onestrain or a mixture of several strains among the microorganisms in thecomposition is applied in the arable soil attached to the seeds ofplants. The microorganisms of the composition are selected fromAzospirillum lipoferum ssp. lip7R 885, Azospirillum amazonense ssp. K21R887, Azospirillum irakense ssp. 5041R 889, Azospirillum brasilense ssp.A41R 879, Azotobacter vinelandii ssp. ESZ 2132, Pseudomonas sp. Szeged344 O.P. 14, Pseudomonas fluorescens var. MOB24, Res24, Bacilluscirculans var. Res. 97, Bacillus megaterium var. Res. 54, Rhizobiummeliloti var. PolRes. 7, Alcaligenes faecalis var. Res36 andPhyllO6-R+32. The strains of the bacteria had been isolated from theenvironment of different plants and dissimilar soils, then have beenmade pesticide resistant. The object of the solution according to thepatent application is other then the antagonism against the pests ofgreen vegetables.

International patent application No. WO9821964 relates to an individualBacillus subtilis strain, which is capable of inhibiting plant pathogenfungi and bacteria. The Bacillus subtilis strain according to the priorart document capable of protecting different plants (fruits, greenvegetables) from the infections caused by the pathogens selected fromthe bacteria and Botrytis, Fusarium, Phytophthora, Pseudomonas, Erwinia,Alternaria, Trichoderma, Monilinia, Puccinia, Rhizoctonia, Phythium ésPlasmopara. The strain may be applied together with pesticides as well,however suffers from the drawback of lacking copper resistance,therefore the application of copper containing pesticides is excluded inthis case.

It can be seen from the above that the solutions according to the stateof the art disclose bacteria relevant from the pest control point ofview, which can be applied together with pesticides, however, they failto disclose a copper resistant bacterium strain, which would enable oneto use further copper containing pesticides in order to achieve moreeffective plant protection. Furthermore, there is need for compositions,with which an effective control of the copper resistant plant pathogenbacteria would be achieved. There is need therefore for novelcompositions against plant pathogen bacteria and fungi, which wouldovercome the drawback of the state of the art, especially theapplication of complicated, multiple step and environmentally stressingpest control technologies. In order to meet these needs we have donesystematic research and development, as a result of which we havecompleted our invention.

DETAILED DESCRIPTION OF OUR INVENTION

Definitions

The term “pest” or “pathogen” as used in the present description means avirus, bacterium or fungus, which lives parasitically in differentliving organisms, and settling and reproducing in the host or its body(e.g. in a human) causes a disease. The measure of the ability toinfect, the pathogenity, may be variable even within one species.

The term “antagonist effect” or “antagonism” as used in the presentdescription means a relationship between microorganisms, in which themembers of one species are killed, deterred, or their reproduction isinhibited by the representatives of another species, e.g. through thechemicals (e.g. antibiotics, extracellular enzymes) produced by them.

The term “pest controlling agent” or “pesticide” as used in the presentdescription means a plant protecting agent (chemicals, other agents anda combination of them), whose application intends to kill, exclude, warnoff, inhibit or any kind of control pathogen organisms, thereby theeffective protection of plants. The pesticide according to the inventionmay be selected by the skilled person on considering health and safetyissues, without undue experimentation.

The term “dose formulation” as used in the present descriptioncharacterises the type of formulation according to the composition,which is determined according to the Pesticide formulation types andinternational coding system catalogue (Crop-Life International TechnicalMonography, No. 2; 5^(th) Edition, 2002). This may be e.g. aqueoussuspension, suspension concentrate, capsulated concentrate, emulsionforming liquid spray, granule, granule dispersible in water,microgranule, water soluble powder, but is not limited thereto. The doseformulation which may be used according to the invention may be selectedby the skilled person without undue experimentation.

The term “copper containing pesticide” as used in the presentdescription means a pest control composition, which contains copper. Themeaning of the copper containing pesticide is not particularly limited,and it may be selected from the group of copper-sulphate,copper-oxyquinolate, copper-oxide, copper-hydroxide andcopper-oxy-chloride. The copper containing pesticide according to theinvention may be selected by the skilled person without undueexperimentation.

The term “excipient” as used in the present description is notparticularly limited, and the excipients may be selected from the groupas follows:

-   a) in case of a liquid formulation e.g. water or an organic solvent    (e.g. xilene, methanol, ethylene-glycol or mineral oil), a    dispersion stabilizator, a surfactant (e.g.    calcium-dodecyl-benzene-sulphonate, polyglycol-ether, etoxylated    alkyl-phenol or alkyl-aryl-sulphonates), optionally waxes,-   b) in case of a granular formulation montmorillonite, bentonite,    wood flour, starch, cellulose and a binder, such as e.g. a mineral    oil, polyvinyl-alcohol or saccharose,-   c) and other in itself known, usual additive and/or excipient.

The excipient according to the invention may be selected by the skilledperson without undue experimentation.

The Discovery According to the Invention

As a result of the systematic experimental work directed to the presentinvention we have surprisingly found that the Bacillus mojavensis R3Bmutant strain

-   a) exerts stronger antagonist effect against pathogens,-   b) carries copper resistance and-   c) induces resistance against pathogens in plants-   unlike/as compared to the state of the art.    Detailed Description Of The Invention

Based on the above, the present invention relates in its first aspect toa biological material for exerting antagonism against vegetablepathogens, which contains Bacillus mojavensis R3B mutant straindeposited under # NCAIM (P) B 001389 according to the Budapest Treaty.The biological material according to the invention is an effectiveantagonist against the pathogens of vegetables, preferably tomato,pepper, lettuce and/or cabbage, in particular against the pathogensselected from the group of Xanthomonas vesicatoria, Pseudomonas syringaeand Clavibacter michiganensis vegetable pathogen bacteria and Pythiumdebaryanum, Phytophthora infestans, Alternaria alternata and Fusariumoxysporum vegetable pathogen fungi.

Although we do not wish to restrict the explanation of the antagonisteffect according to the present invention to one theory, it can be seenthat the unique effect of the biological material according to theinvention against the pathogens is on one hand inhibits the operation ofpathogens in the rhysosphere of the plants and on the other hand, at thesame time it induces resistance in the protected plant against theinhibited pathogens.

In the second aspect, the present invention relates to a compositionthat comprises a culture of the biological material according to theinvention and optionally a copper-containing pesticide, preferablycopper-sulphate, copper-oxyquinolate, copper-oxide, copper-hydroxide orcopper-oxy-chloride, and optionally an excipient.

Based on the above, the copper-containing pesticide according to thepresent invention is not particularly limited, in condition that it doesnot kill the biological material according to the invention, or does notinhibit its operation to an unwanted extent, and such usefulcopper-containing pesticides include copper-sulphate,copper-oxyquinolate, copper-oxide, copper-hydroxide orcopper-oxy-chloride, but are not limited thereto. The pesticideaccording to the invention may be selected by the skilled person withoutundue experimentation.

Based on the above, the excipient according to the present invention isnot particularly limited, in condition that it does not decrease theeffectiveness of the biological material according to the presentinvention as an active agent, or a biological and chemical active agentcombination, and the applicable excipients include without limitationthe following:

-   -   a) in case of a liquid formulation e.g. water or an organic        solvent (e.g. xilene, methanol, ethylene-glycol or mineral oil),        a dispersion stabilizator, a surfactant (e.g.        calcium-dodecyl-benzene-sulphonate, polyglycol-ether, etoxylated        alkyl-phenol or alkyl-aryl-sulphonates), optionally waxes,    -   b) in case of a granular formulation montmorillonite, bentonite,        wood flour, starch, cellulose and a binder, such as e.g. a        mineral oil, polyvinyl-alcohol or saccharose,    -   c) and other in itself known, usual additive and/or excipient.

The excipient according to the invention may be selected by the skilledperson without undue experimentation.

The dose form according to the present invention is not particularlylimited, provided that it is suitable for the application of thebiological material according to the invention as active agent, or thecomposition containing said biological material according to theinvention to the protected plant or any part thereof. Such applicabledose forms include without limitation the following: aqueous suspension,suspension concentrate, capsulated concentrate, emulsion forming liquidspray, granule, granule dispersible in water, microgranule, watersoluble powder. The dose formulation which may be used according to theinvention may be selected by the skilled person without undueexperimentation.

In its third aspect the invention relates to a process for controllingvegetable pathogens according to which the biological material orcomposition according to the invention is applied to a plant, preferablyto a vegetable, more preferably to tomato, pepper, lettuce and/orcabbage. The biological material according to the invention ispreferably applied to the seeds of the protected plant, roots of theprotected plant, stem of the protected plant, leaves of the protectedplant, blooms of the protected plant, the foliage of the protected plantor fruits of the protected plant, is mixed to the irrigation water ofthe plant and/or sprayed to the protected plant.

Finally, the invention relates to the use of the biological material orcomposition according to the invention for the control of pests,preferably for the control of the pests of vegetables, more preferablyfor the control of the pests of tomato, pepper, lettuce and/or cabbage,furthermore, for inducing resistance in said plants against thepathogens according to the present invention.

EXAMPLES

In the following, our invention is further detailed through preparationand working examples, referring to the figures listed below, annexed tothe description.

FIG. 1 shows the copper-ion sensitivity results of the strains showingexcellent antagonist capability in the widest spectrum.

FIG. 2 shows the antagonist activity of 20 bacterium strains as afunction of the copper concentration.

FIG. 3 shows the change of the antagonist effect against fungi as afunction of the copper concentration, an average of 20 strains.

FIG. 4 shows the thin layer chromatograpy of the antibiotics spectrumsecreted by the Bacillus mojavensis B5 strain.

FIG. 5 shows the antibiotics production kinetics of the Bacillusmojavensis B5 strain in a fermentor.

FIG. 6 shows the ability of the Bacillus mojavensis B5 and thecopper-resistant mutants made therefrom to produce extracellularprotease.

FIG. 7 shows the inhibition of Pseudomonas syringae by the a Bacillusmojavensis B5 and the copper-resistant mutants made therefrom.

THE ISOLATION OF BACILLUS MOJAVENSIS B5 STRAIN

In the course of our experiments we have tested the antagonist abilityof 82 endophyte bacterium strains and 44 bacterium strains isolated fromthe rhysosphere, then we have selected the best 20 antagonist strains,and their antagonist abilities have been tested in the presence ofcopper. Among the best antagonist 10 copper-resistant Bacillus strains,the Bacillus mojavensis B5 strain possessed the best antagonistfeatures, especially the R3B copper-resistant mutant strain, which isthe subject matter of our claim for the protection.

The Isolation of Dominant Bacterium Strains from the Rhysosphere andRoots of the Produced Plants, and Testing of their Antagonism

The isolations were made from the root surface and rhizomes of differenttomato and pepper species on bacterium selective culturing medium. 10-10strains were isolated from the dominant colony types in case of eachtested sample. The best antagonists were selected against Pseudomonassyringae, Xanthomonas vesicatoria, Erwinia carotovora, illetvePhytophthora infestans, Sclerotinia sclerotiorum, Alternaria solani andBotrytis cinerea with preliminary antagonism tests on culturing plates.These were identified on species level by partial sequencing their 16SRNS gene, in order to exclude the plant pathogens from the furtherexaminations. The efficacy of the non pathogenic strains was testedagainst another plant pathogenic bacteria and fungi. 40 of the bestantagonists were selected for the in vivo plant treatment examinations.

Isolation and Testing of the Endophyte Bacteria

82 endophyte bacterium strains were isolated from the roots and seeds ofdifferent vegetables (parsley, carrot, tomato, pepper, white cabbage,lettuce, spring onion, cucumber). Their ability to antagonize the plantpathogen Fusarium oxysporum, Rhizoctonia solani; Xanthomonas campestrispv vesicatoria, Erwinia carotovora and Pseudomonas syringae strains wastested by in vitro antagonism tests. The antagonist potential of thebest 10 strains was tested against the plant pathogen fungi Phytophtorainfestans, Botrytis cinerea, Sclerotinia sclerotiorum and Alternariatenuis as well. Furthermore, the effect of pH on the growth was tested:it was determined, which strains can grow at pH=5.5. This has importancein their applicability in more acidic soils. Preliminary coppertolerance test were made with the strains (this is important for thereason of their applicability in the integrated pest control) withculturing media containing 25 and 50 microgram/ml CuSO₄, and found that71 and 49 strains grew in the tested values. To determine the strains,the 16S RNS gene was amplified by PCR (primers: Eub8F és Eub534R),sequenced, then compared with the databases to exclude the plant orhuman pathogen strains. 10 good antagonist bacteria were used for theplant tests out of the 82 original endophyte bacteria.

The Examination of the Bacterium Strains Isolated from the Rhyzosphere

Samples were taken from a greenhouse, from the rock wool of tomatoplants grown in a hydroponic system, and 39 strains were isolated on aculture medium buffered to pH=5.5. Their antagonism was tested againstFusarium oxysporum, Phytophtora infestans, Botrytis cinerea, Sclerotiniasclerotiorum, Alternaria tenuis, Clavibacter michiganense, Xanthomonascampestris pv vesicatoria and Pseudomonas syringae.

From 10 samples originating from arable soil 44 strains were isolated ona pH=5.5 culture medium, 28 strains with the ability to grow in coldconditions, and 27 strains on a Pseudomonas selective culturing mediumusing Bacillus strain selective methods. These strains were testedagainst Clavibacter michiganense, Xanthomonas campestris pv vesicatoriaand Pseudomonas syringae by in vitro antagonism tests. To determine thestrains, the 16S RNS gene was amplified by PCR (primers: Eub8F ésEub534R), sequenced, then compared with the databases to exclude theplant or human pathogen strains.

The Preparation of Copper-Resistant Mutants from the Bacterium StrainsShowing Excellent Antagonism

The copper-ion sensitivity of the strains showing excellent antagonismin the widest spectrum was determined by culturing medium dilutionmethod (FIG. 2). Then strains were prepared with a copper-ion toleranceexceeding 200 μg/ml by selection for spontaneous copper-resistance.

The test was made with 20 pre-selected strains: B2, B5, B7, B12, B14,B19, B23, B40, B52, B60, B73, B83, B198, B208, B209, B212, B215, B218,B219, B221. The strains were maintained on peptone culturing medium at+5° C. temperature, for prolonged shelf life on YDC culturing medium,also at +5° C. temperature. The tests, as the majority of the followingtests, were made on peptone culturing medium or liquid. The coppertolerance of the strains was tested on a culturing medium containing 10,20, 40, 100 and 200 ppm copper. The copper was admixed to the culturingmedium in the form of copper-sulphate, starting from 10000 ppm stocksolution, calculating the concentration for the copper active agent. Thefresh culture of the bacteria was used to prepare a 10⁶/ml suspension,and 100 μl of this was transferred into 9 cm Petri dishes. Theevaluation was made continuously, 2-3 days afterwards. In the test madein the culturing liquid 10 μl of the bacterium suspension with the sameconcentration was added to 10 ml culturing liquid. The evaluation wasmade 2 days afterward, by measuring of the optical density (660 nm), andconfirmed by Bürker camera. The incubation was done in each case at atemperature of 22-25° C., in dark.

All 20 strains grew both on the culturing medium and in the culturingliquid even with 200 ppm copper concentration, and no significantdifference was revealed as compared to the growth at lower copperconcentration.

In the following tests the copper concentration of the culturing mediumwas increased to 400 and 800 ppm, respectively. At 400 ppm the B5, B198,B208, B219 és B221 strains showed no growth anymore, the others stillgrew in a small compass. The 800 ppm concentration resulted in completeinhibition. At the same time, transferring to a culturing medium of 400,then 800 ppm copper concentration, colonies from each strain wereisolated which were able to grow in such a high concentration, and theycould be maintained in a culturing medium with said copperconcentration. As the 400 and in particular 800 ppm copper concentrationis higher than the value necessary to inhibit the growth of plantpathogen fungi and bacteria, further, which may appear in soils, it wasnot reasonable to isolate strains tolerant with even higher copperconcentration. The copper tolerant strains were maintained on aculturing medium containing 800 ppm copper at +5° C. temperature.

The Antagonism Against Plant Pathogens, the Effect of Copper on theAntagonism

The tests were made with the above 20 bacterium strains, against thefollowing plant pathogens:

-   Pseudomonas syringae (5 strains)-   Pythium ultimum-   Phytophthora infestans (3 strains)-   Sclerotinia sclerotiorum-   Fusarium oxysporum fsp. lycopesici-   Alternaria solani-   Rhizoctonia solani

The antagonism tests were made on a peptone agar, on PDA, which is moresuitable for fungi, and in case of Phytophthora strains on pea agar,supplementing the culturing medium with 0, 50, 100 és 200 ppm copper.The antagonist bacterium strains were inoculated starting from a freshculture, using a 5 mm diameter filter paper disc, into one third of the9 cm culturing medium disc, at the same time, in front of them into thesecond third of the culturing medium disc the plant pathogen Pseudomonasstrains were inoculated by a similar method, furthermore, the fungi wereinoculated using a 5 mm diameter mycelium disc cut off the freshculture. The incubation took place in each case at a temperature of22-25° C., in dark.

The evaluation was started 2 days afterwards, and was made continuously,measuring the appeared inhibition zones. The evaluation of the resultswas done by one and two factor analysis of varience.

From the results in can be seen that there were significant differencesbetween the antagonist bacterium strains regarding the antagonism showedagainst the fungi pathogens (Pythium and genuine fungi), however, in theculturing medium containing copper this difference was insignificant(p=0.05). At the same time the difference grew further upon the additionof 50 ppm copper, and 100 and 200 ppm resulted in significant differencebetween the strains (FIG. 2).

The addition of copper slightly increased the antagonist activity,however, the difference on average of five test fungi (P. ultimum, S.sclerotiorum, A. solani, F. oxysporum és R. solani) was significant onlyat 200 ppm copper concentration (FIG. 3). At the same time, it is worthmentioning that this concentration significantly increased the growth ofthe test fungi, and made it difficult to evaluate the results. In casesof B2, B5, B7, B12, B19, B23, B40, B52, B60, B73, B83, B198, B208, B209,B219 and B221 strains a definitely stronger antagonist effect wasexperienced within such circumstances, however, at lower concentrations(50 és 100 ppm) this phenomenon was seen only with the B208, B219 ésB221 strains (FIG. 2).

Considering the average sensitivity of the above-mentioned five testfungi against the antagonists, there were of course significantdifferences, and these differences were significant at every copperconcentration, and even without copper. F. oxysporum and R. solani wereproved to be the least sensitive, in these cases the sensitivity was noteven increased by the elevation of the copper concentration. A. solaniwas moderately sensitive as well, but the copper concentration of 200ppm resulted in a significant increase in the sensitivity. P. ultimumand the tested S. sclerotinia strains were more sensitive, especiallywith high copper concentration.

The reaction of the tested plant pathogen P. syringae bacteria wassignificantly different from the pathogen fungi. There was not anysignificant difference in the effectiveness of the 20 antagonistbacteria considering the average of the 5 P. syringae strains.Similarly, there was no significant difference between the sensitivityof the different P. syringae strains. Unlike the experiences with theplant pathogen fungi, the copper content of the culturing mediumdefinitely decreased the effectiveness of the antagonists. Thedifference experienced was significant only at 100 and 200 ppm copperconcentration. (The copper sensitivity of the plant pathogen P. syringaestrains was approximately the same as that of the 20 antagonistbacteria: all increased at 200 ppm concentration, and none of them at400 ppm.)

The Phytophthora infestans strains involved in the test were moresensitive to the copper ions as compared with the other pathogens, theydid not show growth anymore at 100 ppm concentration, therefore thetests were made only at 50 ppm concentration. There was significantdifference between the efficacies of the antagonist bacteria, however,the presence of copper did not result in significant change (it wassimilar to the other tested fungi, where there was no clear change inthe character of the antagonist activity at 50 ppm copperconcentration).

As to the influence of the copper ions on the efficacy of theantagonists, it was established that the enhancement of the efficacythat may be used in the further tests was experienced only in cases ofplant pathogen fungi and only at a relatively high (200 ppm) copper ionconcentration.

Greenhouse and Field Tests with the Promising Bacterium Strains

Greenhouse and field treatment tests were made with the copper-resistantstrains proven to be the best antagonist. The tests aimed at clarifyingthe rhysosphere tolerance of the anatagonist strains in tomato andpepper culture (culturing methods using soil or using no soil), and inother vegetable cultures (cabbage, lettuce), and especially the veryimportant question of how the plant tolerates the treatment with thebacterium. Using quantitative culturing from the rhizomes of the treatedplants it was clarified if the bacterium strain is incorporated in theplant, if it makes colonies endogenously without adversely affecting thedevelopment of the plant. The tests were run with 20 strains possessingexcellent in vitro antagonism spectrum.

The tests were made in two periods, using 2×10 bacterium strains. Thestrains to be tested (members of the Bacillus, Pseudomonas, Pantoeagenus) were diluted to the required concentration, and the productionboxes and the rock wool sowing platform were irrigated with thissolution immediately after sowing. The further tests were made withyoung plants already having leaves. The plant samples were collected attwo dates, where the leaves of the selected plants were counted, and theplants were collected without their roots and their fresh green mass wasweighed. The tests were made with pepper and tomato species, in soil androck wood production system, considering the irrigation with threedifferent bacterium concentrations. In cases of cabbage and lettuce thetests were set only to the soil production method. All tests were madein four sets, in random block arrangements.

10 Bacillus strains have proven to be the best.

Greenhouse and Field Tests II. Testing of the Ability to InduceResistance

Regarding the antagonist bacterium component according to our plannedcompositions it is an important requirement that it should provideprotection against the bacterial and fungal pathogens not only at therhizosphere, but also at the organs of the plants above the soil throughthe activation of the inducible resistance mechanisms of the plant. Thetests were made with tomato and pepper. The roots of the young plantswere treated with the suspension of the antagonist bacterium strains,and after planting, when different intervals in time have passed,artificial infections were provoked on the leaves with a cell andconidium suspension of the plant pathogen bacteria and fungi.Furthermore, it was evaluated how the level of the chemicals playingimportant role in the induced resistance changes in the treated plantsas compared with the control (reference) plants.

Our excellent antagonist B5 Bacillus strain had the ability to inducethe self-protection mechanism of the plant [Systemic Acquired,Resistance, (SAR)]. The R3B copper resistant mutant spontaneouslyappearing after transferring of the Bacillus mojavensis B5 strain onto aculturing medium containing 400 ng/ml copper-sulphate was proven to bethe best. This strain possesses wide spectrum and excellent antagonistabilities probably for the reason of secreting in large amount anantifungal depsi-peptide antibiotics, fengycin (FIG. 5), and theconstitutive chimotripsin type protease, which enhances the effectthrough synergism (FIG. 6).

Evaluation of the Antibiotics and Chimotripsin Production of theBacillus mojavensis B5 Strain and the Copper Resistant Mutants MadeTherefrom

The strains were maintained by weekly cross inoculation, on YEGculturing plates (0.2% glucose, 0.2% yeast extract, 2% baktoagar). Forthe antibiotics production tests mainly minimal culturing solution wasused suitably changing the used carbon and nitrogen source, furthermorethe concentration of the two trace elements (iron and copper). Thisculturing solution, unlike e.g. a yeast extract based culturingsolution, does not contain amino acids and peptides, thus the analysisand purification of the antibiotics mixtures secreted into thefermenting liquid can be made more easily.

Based on the preliminary production experiences, the following GGMculturing solution was used:

Glucose  1% Glutaminic acid Na salt 0.5% KH₂PO₄ 0.1% K₂HPO₄ 0.1% MgSO₄ ×7H₂O 0.05%  KCl 0.1% FeSO₄ × 7H₂O 10 mg/l CuSO₄ × 5H₂O  1 mg/l20 ml culturing solution was measured into 50 ml Erlenmeyer flasks.After inoculation the culturing took place on an orbital shaking devicefor 6 days, at 180 rpm and 25° C. temperature.

The cell density of the cultures was determined by measuring theabsorption at 620 nm. In case of the Bacillus strains a 0.1 OD value isequivalent with a 10⁷ cell/ml concentration. Thereafter the bacteriumcells were settled by centrifuging at 8000 G for 10 minutes, and thesupernatant was transferred to a beaker, then the pH of the fermentedliquids was set to 2 using 10% hydrochloric acid (0.4 ml to 20 mlfermented liquid). The precipitated fermented liquids were incubated at5° C. temperature overnight, to complete the precipitation. Then theprecipitate was settled by centrifugation, and dissolved in 1 ml 96%ethanol.

The Quantitative Determination of the Antibiotics from the EthanolPreparations

The fengycin molecule contains 2 tyrosine molecules, which however showsa strong absorbance at 280 nm. Thus, the antibiotics content other thansurfactin of the antibiotics preparations may be estimated by measuringat 280 nm. Thus, all OD of the ethanol preparations diluted to 10 timesof their original concentration was measured at 280 nm.

Thin Layer Chromatograpy (TLC) of the Secreted Antibiotics

In our preliminary studies, as well as in the tests of the novel strainsin the thin layer chomatograpic analysis of the strains a glutaminicacid/glucose culturing solution was used, as in cases of most strainsthis provided the sufficient yield of antibiotics. FIG. 4 shows theresults of the TLC analysis of the fermented liquid of the B5 strain.

In the course of the pest control application it is extremely importantto use the suitable culturing medium when preparing the cultures. Theresults prove that the above-mentioned glutaminic acid/glucose culturingsolution should be used, as the culture may be diluted even to 10-20times of its original volume, and even in this case the fengycinconcentration/ml is 10-12 mg/l, which achieves a complete inhibition ofthe majority of the plant pathogen fungi, and is sufficient to activatethe induced resistance mechanism in the treated plant.

The Antagonist Ability of the B. mojavensis B5 Strain and the CopperResistant Mutants Spontaneously Produced Therefrom

The strains were cultured on a yeast extract culturing medium for 48hours. The inhibition of Pseudomonas syringae, Clavibacter michiganensiand Xanthomonas campestris by the B5 strain and its copper resistantmutants was tested. It can be seen in the table that the best inhibitingeffect is possessed by the R3B copper resistant mutant.

Inhibition zone (mm) B-5 R1B R2 R3A R3B R4 R6 Pseudomonas syringae 3.02.0 4.0 3.0 5.0 2.0 3.0 Clavibacter michiganensis 6.0 2.0 3.0 6.0 7.05.0 4.0 Xanthomonas campestris 7.0 5.0 6.0 7.0 7.0 7.0 8.0

The copper-resistant B5 strains' inhibition effects on Fusariumosysporum was compared to the parent strain. The table shows that inthis case also the R3B copper resistant mutant possesses the bestefficacy. Therefore, we seek protection for this strain.

Inhibition zone (mm) B-5 R1B R2 R3A R3B R4 R6 Fusarium oxysporum 3.5 3.53.0 4.0 4.0 3.5 4.5

INDUSTRIAL APPLICABILITY OF THE INVENTION

It is a benefit of the present invention that the isolated Bacillusmojavensis R3B mutant strain induces a strong antagonist effent againstpathogens, and also carries copper-resistance and has the ability toactivate an induced resistance in plants against the pathogens. Due toits copper-resistant feature, it may be used together with coppercontaining pesticides, thus may effect an integrated pest control.

What is claimed is:
 1. A biological material for exerting antagonismagainst vegetable pathogens, which contains Bacillus mojavensis R3Bmutant strain deposited under # NCAIM (P) B 001389 according to theBudapest Treaty.
 2. A process for controlling pathogens, said processcomprising applying the biological material according to claim 1 to aplant.
 3. The process according to claim 2, said process comprisingapplying the biological material to a) seeds of the plant, b) roots ofthe plant, c) stems of the plant, or d) leaves, flowers, foliage orfruits of the plant, by mixing the biological material with irrigationwater of the plant and/or spraying the biological material to the plant.4. The process of claim 2, wherein the pathogens are pathogens oftomato, pepper, lettuce or cabbage.
 5. The process of claim 2, whereinthe plant is a vegetable.
 6. The process of claim 2, wherein the plantis tomato, pepper, lettuce or cabbage.
 7. The process according to claim2, wherein the pathogens are pathogens of green vegetables.
 8. Theprocess according to claim 2, wherein the pathogens are selected fromthe group consisting of Xanthomonas vesicatoria, Pseudomonas syringaeand Clavibacter michiganensis plant pathogen bacteria and Pythiumdebaryanum, Phytophthora infestans, Alternaria alternate and Fusariumoxysporum plant pathogen fungi.
 9. A pesticide composition, whichcontains a culture of the biological material according to claim 1, andcontains a copper-containing pesticide, and optionally an excipient. 10.The composition according to claim 9, which is in an aqueous suspension,suspension concentrate, capsulated concentrate, emulsion forming liquidspray, granule, granule dispersible in water, microgranule or watersoluble powder dose formulation.
 11. The pesticide composition of claim9, wherein the copper-containing pesticide is selected from the groupconsisting of copper-sulphate, copper-oxyquinolate, copper-oxide,copper-hydroxide and copper-oxy-chloride.
 12. A process for controllingpathogens, said process comprising applying the pesticide composition ofclaim 9 to a plant.
 13. The process according to claim 12, said processcomprising applying the pesticide composition to a) seeds of the plant,b) roots of the plant, c) stems of the plant, or d) leaves, flowers,foliage or fruits of the plant, by mixing the biological material withirrigation water of the plant and/or spraying the biological material tothe plant.
 14. The process of claim 12, wherein the plant is avegetable.
 15. The process of claim 12, wherein the plant is tomato,pepper, lettuce or cabbage.
 16. The process according to claim 12,wherein the pathogens are pathogens of green vegetables.
 17. The processof claim 12, wherein the pathogens are pathogens of tomato, pepper,lettuce or cabbage.
 18. The process according to claim 12, wherein thepathogens are selected from the group consisting of Xanthomonasvesicatoria, Pseudomonas syringae and Clavibacter michiganensis plantpathogen bacteria and Pythium debaryanum, Phytophthora infestans,Alternaria alternate and Fusarium oxysporum plant pathogen fungi.